Our objective is to develop intravenously delivered recombinant Muscle Blind 1 (MBNL1) for patients with Myotonic Dystrophy Type 1 (DM1). DM1 is the most common muscular dystrophy of adulthood and is caused by a large CTG expansion in the 3'untranslated region of the DMPK gene (1-3). Although the function of DMPK and the neighboring Six5 gene are negatively affected by the CTG expansion, the lack of DMPK activity does not fully account for the observed phenotype in DM1;including muscle wasting and myotonia, insulin resistance, testicular atrophy, cutaneous tumors cardiac arrhythmia and cognition defects (4- 8). Subsequent studies have shown that the large CUG expansion within the transcribed DMPK mRNA avidly binds, sequesters and inactivates the MBNL1 protein, an mRNA splicing factor that removes fetal exons from mRNA templates (9- 13). The inactivation of MBNL1 by polyCUG expansions or through genetic ablation of MBNL1 results in the inappropriate expression of fetal proteins in adult differentiated tissues (9, 14- 17). Though sequestration of MBNL1 clearly cannot provide a unitary explanation for DM1, evidence from transgenic mouse and fly models, and studies of patient-derived cells, supports the idea that symptoms of DM1 are partly determined by the stoichiometry of CUG expansion RNA in relation to ambient supplies of MBNL1 protein (18, 19, 20). The consensus in the DM1 community is that any approach that restores the availability of MBNL1 proteins for mRNA splicing would constitute a therapy for DM1 (21, 20). Treatment options being considered for DM1 include [1] small-molecule induced overexpression of endogenous MBNL1, [2] disruption of the polyCUG- MBNL1 association through small-molecule and nucleotide-based therapies, [3] transgenic overexpression of MBNL1 via gene therapy, and [4] direct intravenous application of exogenous MBNL1. 3E10 is a murine-derived monoclonal antibody that penetrates living cells and localizes to the cell nucleus without apparent injury to target cells (22, 23). A single chain Fv fragment of 3E10 (Fv3E10) possesses all the cell penetrating capabilities of the original monoclonal antibody and proteins such as catalase, dystrophin, HSP70 and p53 retain their activity following conjugation to Fv3E10 (24-27). The ENT2 nucleotide scavenger transporter is enriched in skeletal muscle and cancer cells and mediates the cell-penetrating ability of Fv3E10 and Fv3E10 conjugates (28). Given the affinity of Fv3E10 for skeletal muscle and the ability of Fv3E10 conjugates to maintain their respective activities, Fv3E10-based therapies would represent a versatile approach to treat many myopathies, including DM1, DM2, Duchenne muscular dystrophy and Emery-Dreifuss syndrome. We seek funding for two years to translate 3E10 and MBNL1 into a commercially viable product for DM1. We will chemically or genetically conjugate 3E10 to MBNL1, test the purified material in DM1 cell lines, inject the purified material into DM1 mouse models and evaluate any correction of the disease endpoints. To execute this proposal we have gathered the appropriate technology, the commitment from the biotechnology industry, and the expertise and resources of the DM1 scientific and patient advocacy community. Successful conclusion of this proposal will justify further product optimization, including examination of truncated and/or humanized 3E10-MBNL1 and determination of the optimal manufacturing process. The final product concept will undergo further efficacy, pharmacology and toxicology studies, scaled-up GLP production, additional pre- IND pharmacology and toxicology studies, and upon FDA approval the development of phase 1 and 2 clinical trials. PUBLIC HEALTH RELEVANCE: Myotonic dystrophy is the most common muscular dystrophy of adults for which there are no effective therapies. We will test if a muscle-targeted Muscleblind protein therapy will alleviate the spliceopathy in DM1 mouse models.